Excitation-contraction (EC) coupling in skeletal muscle refers to the process by which depolarization of the muscle plasma membrane leads to release of calcium ions into the cytoplasm from the sarcoplasmic reticulum (SR). EC coupling is performed by an array of voltage-sensor proteins (dihydropyridine receptors (DHPRs) in the plasma membrane/transverse tubule system that physically interact with and control an array of SR- embedded calcium-release channels (ryanodine receptors (RyRs), in conjunction with numerous other proteins. This assemblage of proteins (termed a couplon) occurs at regular intervals in muscle, and elucidating its structural architecture, ultimately at the atomic level, is essential to understanding the molecular mechanism of EC coupling in healthy and diseased muscle. Current models of the couplon structure are based largely upon visual interpretation of classical electron microscopy (EM) images, and are highly schematized and qualitative. The first aim of this proposal is to use the latest quantitative cryo-EM and tomographic reconstruction techniques to determine actual mass density distributions of the proteins and membranes comprising the couplon. Focused-ion-beam milling, a new technique for cutting sections of frozen-hydrated tissue that is being developed at our Center, will be used to prepare muscle for cryo-EM; the micrographs and tomograms obtained using this new technique will be compared to those obtained by the more standard technique of cryo-ultramicrotomy. Once optimal methodology is established, hundreds of tomograms will be obtained, and RyRs, which, being large 2.3 MDa complexes, are easily detected in tomograms, will be computationally extracted, classified, and averaged to maximize the contrast and resolution (the goal is 3-4.5 nm). The averaged RyRs are expected to resolve RyR-associated proteins, such as the RyR-DHPR complex which has never been observed. For aim 2, in vitro assembly experiments will be done to make complexes of purified RyR with its natural ligands, such as components of the DHPR and the regulatory protein calmodulin. Cryo-EM and 3D single-particle image analysis will be applied to these complexes to determine in more detail the nature of the interactions and dynamics of RyR and its binding partners.